We thank the Central Animal Facility, IISc for supplying us with mice for research. This study was funded by grants to DpN from the Department of Biotechnology and Science and Engineering Research Board, Government of India. The infrastructural support from the DBT-IISc program and DST-FIST grants are greatly acknowledged. We thank all previous and current members of the DpN lab for their support.

All experiments using Salmonella Typhimurium were conducted in Bio Safety Level 2 (BSL-2) facilities. Care must be taken to use proper personal protective equipment (PPE), ensure safety, and follow standard BSL-2 biohazard disposal methods. All mice experiments were conducted following guidelines stated by the Institutional Animal Ethics Committee, IISc. Mice were bred and maintained at the Central Animal Facility of IISc (Registration number: 48/1999/CPCSEA, dated 1/3/1999), approved by the Ministry of Environment and Forest, Government of India. The experimental protocols were approved by the Committee for Purpose and Control and Supervision of Experiments on Animals with the approved permit number CAF/Ethics/797/2020.
BSL2 definition: A BSL2 rating represents that the biohazardous agents pose a moderate threat to the environment and laboratory staff5.
1. Culture preparation of Salmonella&#160;Typhimurium&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
<ol>
	<li>Add 100 &#181;L of Salmonella Typhimurium NCTC 12023 glycerol stock to 3 mL of Luria Bertani (LB) broth. Incubate the culture at 160 rpm at 37 &#176;C overnight.</li>
	<li>Streak 50 &#181;L of the overnight grown culture in LB broth onto a Salmonella Shigella (SS) agar plate and incubate at 37 &#176;C for ~12 h. Store the SS agar plate with the bacterial colonies at 4 &#176;C for several days before the in vivo infection experiment.</li>
	<li>Pick a single colony from the streaked SS agar plate using a microtip. Eject the microtip into 3 mL of LB broth and culture at 160 rpm at 37 &#176;C overnight.</li>
	<li>Add 0.1 mL of the bacterial culture to 50 mL of LB broth and incubate at 37 &#176;C in a shaker incubator at 160 rpm for 3-4 h to reach the logarithmic phase of growth. Dilute the culture by a factor of 2 using LB broth. 
	NOTE: During the logarithmic phase, bacterial cells are at their best health and are actively dividing.</li>
	<li>Measure the optical density (OD) of the culture at 600 nm wavelength of light in a spectrophotometer or microplate reader. Once the OD reaches 1.0, make two aliquots of 1 mL of culture in 1.5 mL microfuge tubes.</li>
	<li>Centrifuge the tubes at 7,750 &#215; g for 15 min. Discard the supernatant and wash the pellet with 1 mL of 1x PBS 2x. Centrifuge the tubes at 7,750 &#215; g for 15 min.</li>
	<li>Resuspend the pellet in 0.5 mL of 1x PBS in two different 1.5 mL microfuge tubes. Combine the suspensions from both the tubes into one 1.5 mL tube now containing ~2 &#215; 108 colony-forming units (CFU)/mL.</li>
	<li>Prepare a bacterial cell suspension of 1 &#215; 106 CFU/mL by diluting this stock solution. 
	&#8203;CAUTION: Optimize the CFU corresponding to OD under specific laboratory conditions to determine the CFU for OD 1.0 before initiating the experiments.</li>
</ol>
2. Mice and infections
<ol>
	<li>House 8-10-week-old male C57BL/6 mice weighing ~20 g in the clean-air room of the animal facility for several days for acclimation.</li>
	<li>On the day of infection, hold the mouse with one hand, wipe the abdominal skin with 70% ethanol, and spread the hind legs for better accessibility of the abdominal wall.</li>
	<li>Inject 0.5 mL of 1 &#215; 106 CFU/mL bacterial suspension intraperitoneally with the help of a 1 mL syringe. Therefore, each mouse receives 5 &#215; 105 CFU. Control, uninfected mice receive 0.5 mL of PBS alone. Post infection, plate the culture to check the actual CFU injected, which may vary from 0.2-0.8 million CFU/0.5 mL.</li>
	<li>Put the mice back in the cages as assigned.</li>
	<li>Sacrifice the mice using CO2 asphyxiation ~12-18 h post infection for the best response. Usually, all infected mice die within 96 h. Under some experimental interventions, some mice may survive. Euthanize these mice after 96 h. Also, euthanize any mouse with a body temperature below 33.2 &#176;C and acute distress at 96 h as a humane endpoint. 
	&#8203;NOTE: In this model, some mice may start dying after 12 h of Salmonella injection. Therefore, plan experiments properly involving multiple time points.</li>
</ol>
3. CFU assessment of organs
<ol>
	<li>Sacrifice the infected mouse by CO2 asphyxiation, and wipe the abdomen with a piece of cotton dipped in 70% ethanol. Cut open the abdominal skin. Refer to the article by Ray and Dittel for a video protocol on how to collect peritoneal lavage fluid6. Cut open the peritoneal cavity and collect the organs of interest in microfuge tubes. Additionally, as the blood in mice with sepsis gets coagulated fast and the amounts are low, collect it quickly after sacrificing. 
	NOTE: This video demonstrates the enumeration of organ CFU from the liver as the liver undergoes extensive histopathological damage in this model of sepsis.</li>
	<li>Cut a small piece of the liver and place it in a microfuge tube. 
	NOTE: This can be stored on ice for a maximum of 2-3 h before proceeding to the next step.</li>
	<li>Weigh and transfer the pieces to microcentrifuge tubes. Preferably, cut pieces weighing ~10-15 mg for proper homogenization. Use entire organs in the case of smaller ones such as the mesenteric lymph nodes (MLNs) or thymus.</li>
	<li>Add 0.5 mL of 1x PBS to the tube and homogenize the organs using a hand homogenizer. Make sure that the organs are completely homogenized. Make up the volume to 1 mL by adding 0.5 mL of 1x PBS.</li>
	<li>Centrifuge the tubes at 200 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Collect the supernatant into fresh microfuge tubes and prepare dilutions of 1 &#215; 10&#8722;1 and 10&#8722;2 in a 96-well plate.</li>
	<li>Spread 50 &#181;L of the diluent onto fresh SS agar plates and incubate the plates at 37 &#176;C for 12 h.</li>
	<li>Count the number of colonies that appear in each condition and normalize the data with the organ weight using Equation (1): 
	CFU/mg = <img alt="Equation 1" src="/files/ftp_upload/63695/63695eq01.jpg" style="margin: auto;" /> (1) 
	NOTE: The number 20 is used in the formula to convert the colonies per plate to CFU/mL. This number is arrived at by dividing 1 mL by the amount of a given volume of the culture plated-in this case, 50 &#181;L. 
	For example, if 100 colonies are found in an SS agar plate, where 50 &#181;L of a 1 &#215; 10-1 dilution of a homogenized organ weighing 10 mg is spread, then 
	CFU/mg = <img alt="Equation 2" src="/files/ftp_upload/63695/63695eq02.jpg" style="margin: auto;" /></li>
</ol>
4. Flow cytometric analysis of various immune cell populations in peritoneal exudate
<ol>
	<li>Collect the peritoneal cells as described previously by Ray and Dittel6.</li>
	<li>Resuspend the cell pellet from peritoneal lavage fluid in 1 mL of RPMI supplemented with 10% fetal bovine serum (FBS). Enumerate the total cell numbers in the peritoneal lavage using a hemocytometer. Adjust the cell number so that every tube receives 0.2-0.5 million cells. 
	NOTE: Peritoneal lavage in mice with sepsis may contain RBCs, which probably appear due to hemorrhage. Be careful to exclude RBCs while counting peritoneal cells. In the brightfield microscope, RBCs appear much smaller than the immune cells. These appear as flat disks or doughnuts, round, with an indentation in the center, but not hollow. A step to lyse RBCs may be added7.</li>
	<li>Spin the cells down at 200 &#215; g at 4 &#176;C for 10 min, discard the supernatant, and wash the cells 1x with 1x cold PBS. Centrifuge the cells at 200 &#215; g at 4 &#176;C for 10 min.</li>
	<li>Block the Fc receptors on PECs using FcR blocker (1:400 dilution), prepared in blocking buffer consisting of 5% FBS and 0.02% sodium azide in PBS. Incubate on ice for 15 min.</li>
	<li>Centrifuge the cells at 200 &#215; g at 4 &#176;C for 10 min. Discard the supernatant. Dilute the fluorochrome-conjugated antibodies of interest in blocking buffer. Use a 1:500 dilution of anti-mouse LY6G to stain the neutrophils. 
	NOTE: Other immune cell populations can also be detected using, for example, anti-mouse B220 for B cells, anti-mouse CD3 for T cells, and anti-mouse F4/80 for macrophages.</li>
	<li>Incubate ~0.2 million cells in 200 &#181;L of the diluted solutions of antibodies in separate tubes. As a negative control, set aside one tube in each fluorochrome type for the unstained control to incubate the cells with 200 &#181;L of blocking buffer without antibody.</li>
	<li>Incubate the samples on ice for 45 min with intermittent tapping every 15 min.</li>
	<li>Centrifuge the cells at 200 &#215; g for 10 min at 4 &#176;C. Discard the supernatant. Fix the cells with 4% paraformaldehyde for ~15 min at room temperature if they need to be be stored for several days.&#160;However, it is best to acquire data from freshly stained samples in a flow cytometer.</li>
	<li>Resuspend the cells in 200 &#181;L of FACS staining buffer (2% FBS in PBS). Acquire the data in a flow cytometer.</li>
</ol>

<ol>
	<li>Hotchkiss, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+and+septic+shock.">Sepsis and septic shock.</a> Nature Reviews Disease Primers. 2 (1), 1-21 (2016).</li><li>Rudd, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional,+and+national+sepsis+incidence+and+mortality,+1990-2017:+Analysis+for+the+Global+Burden+of+Disease+Study.">regional, and national sepsis incidence and mortality, 1990-2017: Analysis for the Global Burden of Disease Study.</a> The Lancet. 395 (10219), 200-211 (2020).</li><li>Vincent, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+study+of+the+prevalence+and+outcomes+of+infection+in+intensive+care+units.">International study of the prevalence and outcomes of infection in intensive care units.</a> JAMA. 302 (21), 2323-2329 (2009).</li><li>Lewis, A. J., Seymour, C. W., Rosengart, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+murine+models+of+sepsis.">Current murine models of sepsis.</a> Surgical Infections. 17 (4), 385-393 (2016).</li><li>Ta, L., Gosa, L., Nathanson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+and+biohazards:+Understanding+biosafety+levels+and+meeting+safety+requirements+of+a+biobank.">Biosafety and biohazards: Understanding biosafety levels and meeting safety requirements of a biobank.</a> Biobanking. 1897, 213-225 (2019).</li><li>Ray, A., Dittel, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Peritoneal+Cavity+Cells.">Isolation of Mouse Peritoneal Cavity Cells.</a> Journal of Visualized Experiments: JoVE. (35), e1488 (2010).</li><li>Liu, X., Quan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+isolation+from+mouse+femur+bone+marrow.">Immune cell isolation from mouse femur bone marrow.</a> Bio-protocol. 5 (20), 1631 (2015).</li><li>Yadav, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+2+enhances+the+survival+of+mice+during+Salmonella+Typhimurium+infection-induced+sepsis+by+increasing+reactive+oxygen+species,+inflammatory+cytokines+and+recruitment+of+neutrophils+to+the+peritoneal+cavity.">Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity.</a> Free Radical Biology &amp; Medicine. 116, 73-87 (2018).</li><li>Verma, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+hemoglobin+is+a+marker+of+systemic+inflammation+in+mouse+models+of+sepsis:+A+Raman+spectroscopic+study.">Cell-free hemoglobin is a marker of systemic inflammation in mouse models of sepsis: A Raman spectroscopic study.</a> Analyst. 146 (12), 4022-4032 (2021).</li><li>Cassado, A. D. A., Lima, M. R. D., Bortoluci, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+mouse+peritoneal+macrophages:+Heterogeneity,+development,+and+function.">Revisiting mouse peritoneal macrophages: Heterogeneity, development, and function.</a> Frontiers in Immunology. 6, 225 (2015).</li><li>Yadav, S., Verma, T., Chattopadhyay, A., Nandi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+pathophysiology+of+sepsis,+an+inflammatory+disorder:+Key+roles+of+oxidative+and+nitrosative+stress.">Factors affecting the pathophysiology of sepsis, an inflammatory disorder: Key roles of oxidative and nitrosative stress.</a> Indian Journal of Inflammation Research. 3 (1), 2 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

This article describes a method of inducing a severe form of bacterial sepsis by intraperitoneal injection of Salmonella Typhimurium. This model is advantageous over others as Salmonella Typhimurium is an intracellular pathogen and, hence, highly pathogenic, mimicking the clinically relevant condition of Gram-negative sepsis. The outcome of peritonitis sepsis in this model is systemic, with 100% mortality within 96 h post infection. Therefore, this model is instrumental in studying the inflammatory and lethal host responses8 and the effects of therapeutic intervention in attenuating sepsis.
In this model system, the cellular composition of the peritoneal cavity changes dramatically to combat the inoculated pathogen. A large number of LY6G+ neutrophils infiltrate the peritoneal cavity, with a simultaneous decrease in F4/80+ macrophages, resulting in the macrophage disappearance reaction (MDR)8,10. Therefore, this model is useful in studying the kinetic changes in immune cell compositions and functions during sepsis, which is still a vastly unexplored area. Salmonella Typhimurium systemically infects, resides in, and proliferates in the organs of mice. The serum proinflammatory cytokines such as TNF-&#945;, IL-6, and IFN-&#947; also increase8. A cascade of proinflammatory responses also leads to hemolysis and coagulation of the blood, which becomes visible upon dissection of the infected mice10,11. The interplay between the host response to this bacterial burden and the damaging effects of the bacteria leads to disfigured tissue architecture, loss of functions, and tissue necrosis, especially in the liver.
Moreover, previous reports from the laboratory have shown that such effects are modulated by the upregulated expression of nitric oxide synthase 2 (NOS2), which leads to increased reactive oxygen species (ROS) and higher levels of proinflammatory cytokines, leading to an enhanced inflammatory response8. This mouse sepsis model is advantageous over others because the induction of sepsis is faster with more lethal effects. It does not require technical expertise to perform the surgery on anesthetized mice, as is the case with the current gold-standard CLP-sepsis mouse model. In the CLP-sepsis model, only a subset of mice develops sepsis, whereas all mice develop sepsis in the model described here.
Moreover, compared to the LPS-induced endotoxemia sepsis model, this model is more clinically relevant for Gram-negative monobacterial sepsis. The model is also useful in understanding the increases in intracellular ROS, proinflammatory cytokine levels, hemolysis, and blood coagulation, all of which happen during sepsis. It is reported that the use of the nitric oxide donor drug DETA-NONOate provides a survival benefit in Nos2 knockout mice with sepsis using this model8. This suggests that this model can be used to identify novel drugs to treat sepsis. Also, this model can be quickly adopted by any laboratory equipped with BSL-2 facilities.
While using this model, precautions must be taken at the level of bacterial cell health and the CFU number to obtain optimal results. One should always use freshly streaked Salmonella colonies from SS agar plates. It is also recommended to optimize the process of culture preparation by finding the OD value in the spectrophotometer corresponding to the mentioned CFU.
The disadvantage of using this model is that the effect of sepsis in mice is intensely lethal within 96 h post infection. This limits the study of host immune responses at late time points. It can be overcome by using a more attenuated bacterial strain of Salmonella Typhimurium.
There are differences between human patients suffering from sepsis and mice models attempting to mimic human sepsis. For example, patients take longer to develop sepsis and are put on supportive interventions and therapeutics. However, sepsis in mouse models develops much faster, and supportive interventions are not done4. However, there are some similarities between sepsis in humans and mice, with patients with sepsis showing higher levels of proinflammatory cytokines, blood coagulation, and hemolysis in clinical settings. All these are mimicked in this mouse model of sepsis. Therefore, some readouts of this model are relevant to clinical scenarios, although a direct correlation may not be advisable.

Sepsis is defined as a dysregulated systemic inflammatory and immune response to microbial invasion or tissue damage, leading to organ injury distant from the site of infection or damage. Septic shock is a subset of sepsis characterized by hypotension persisting during volume resuscitation, with a substantially increased risk of mortality1. The general public has become more aware of this disorder during the COVID-19 pandemic. Despite its high associated mortality, comprehensive epidemiological data on the global burden of sepsis is lacking because of the complexity of its diagnosis. In 2017, there were 48.9 million sepsis incidences and 11 million deaths worldwide, accounting for 19.7% of all global deaths2. Further, a study on the extended prevalence of infection and related sepsis in intensive care unit patients found that 62% of the positive isolates from patients were Gram-negative organisms3.
Initially, the investigations on sepsis focused on delineating microbial pathogenesis. However, understanding the &#34;danger hypothesis&#34;, which dictates how the host distinguishes self and non-self, led to the tilting of the balance of sepsis research toward understanding the host response to an invading pathogen. The widely used mice models of sepsis include the lipopolysaccharide (LPS)-induced endotoxemia model, polymicrobial sepsis models, cecal ligation and puncture (CLP) and colon ascendens stent peritonitis (CASP), and monobacterial infection models4.
We have standardized a mouse model system by inducing peritoneal sepsis using Salmonella Typhimurium. This model is advantageous over others because Salmonella Typhimurium is an intracellular pathogen that mimics the clinically relevant condition of Gram-negative sepsis. The outcome of peritonitis sepsis in this model is systemic, with 100% mortality within 96 h post infection. Therefore, this model is instrumental in studying the inflammatory and lethal host responses. In this model, sepsis is induced by intraperitoneally injecting 0.5 million colony-forming units (CFU) of Salmonella Typhimurium into an 8-10-week-old C57BL/6 mouse. Systemic infection can be confirmed by assessing organ bacterial burden ~16 h post infection. This article demonstrates Salmonella Typhimurium-induced peritonitis sepsis in mice, characterizes the resulting alterations in peritoneal cell composition, and quantifies bacterial burden in different organs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Sterile Syringe with 26 G needle</td>
			<td>Beckton Dickinson, Singapore</td>
			<td>303060</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tube</td>
			<td>Tarsons, USA</td>
			<td>500010</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Sterile Syringe with 21 G needle</td>
			<td>Beckton Dickinson, Spain</td>
			<td>307758</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Flask</td>
			<td>Tarsons, USA</td>
			<td>441150</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Graduated Centrifuge Tube</td>
			<td>Tarsons, USA</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Graduated Centrifuge Tube</td>
			<td>Tarsons, USA</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell spreader</td>
			<td>VWR, USA</td>
			<td>VWRU60828-680</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>HiMedia, Mumbai, India</td>
			<td>TS1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocker</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat anti-mouse Ly6G (Clone 1A8)</td>
			<td>BD Pharmingen</td>
			<td>551460</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand based Homogenizer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (Neubauer counting chamber)</td>
			<td>Rohem, India</td>
			<td>I.S. 10269</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>HiMedia, Mumbai, India</td>
			<td>M1245</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Petriplates</td>
			<td>Tarsons, USA</td>
			<td>460091</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Himedia, Mumbai, India</td>
			<td>AT060-10X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella-Shigella Agar</td>
			<td>HiMedia, Mumbai, India</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Kubota</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD FACSverse</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>N-biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing machine</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing salmonella typhimurium-induced septic peritonitis in mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

A detailed characterization of the host immune response using this particular model is shown in previous publications8,9. A few representative results of the described protocol are depicted in this section. This model aims to induce systemic infection of S. Typhimurium by intraperitoneal injection of the bacterial culture to induce sepsis. To confirm the infection, the lysates of the liver and spleen from septic mice were spread on SS agar plates, and the number of colonies was counted. In Figure 1, the images of SS agar plates indicate the organ CFU burden in the liver and spleen of septic mice. The homogenized organ lysates were spread at a dilution of 1 &#215; 10&#8722;1 and incubated at 37 &#176;C.&#160;The black pigmented S. Typhimurium colonies appeared after ~12 h of incubation at 37 &#176;C. Furthermore, upon infection of mice, the cultures from blood and peritoneal lavage are reported to be positive for bacterial cells8. These results indicated that the intraperitoneally injected bacterial cells disseminated systemically and colonized the internal organs. A part of the plate is shown as a zoomed-in inset to highlight the colonies. The pathogen successfully disseminated systemically and colonized the internal organs.
Figure 2 shows the sera isolated from healthy and septic mice. The volume of collectible blood through cardiac puncture from septic mice is usually 100-200 &#181;L, which is lower than the amount from healthy mice, where ~500 &#181;L of blood can be obtained. As a result, the serum volume isolated from septic mouse blood is low. This phenomenon happens because of the increased coagulation of the blood in septic mice. Moreover, the sera from septic mice show distinct red coloration, indicating the occurrence of extensive hemolysis8,9.
As shown in Figure 3, flow cytometry-based immunophenotyping of peritoneal exudate cells was performed to assess the infiltration of neutrophils in the peritoneal cavity of healthy and septic mice. As neutrophils express LY6G protein on the cell surface, FITC-tagged anti-LY6G antibody was used to stain the neutrophil cell population. These images represent data from one healthy and two infected mice. After data acquisition, the cells were gated to include only singlets in an FSC-A versus SSC-A plot. Then, the histogram plots and dot-plot were drawn. Here, the septic mice showed increased infiltration of neutrophils in the peritoneal cavity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63695/63695fig01.jpg" /> 
Figure 1: Visualization of organ CFU post infection. Organs were homogenized as described in the protocol. The lysate was spread on SS agar plates using a spreader. Images of plates spread with a 1 &#215; 10-1 dilution of homogenized lysates of (A) liver and (B) spleen. A part of the plate is shown as a zoomed inset to highlight the colonies. Inset magnification = 13x (area). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63695/63695fig02.jpg" /> 
Figure 2: Enhanced hemolysis in septic mice. The blood was collected in microcentrifuge tubes and left undisturbed at room temperature for 30 min. The clot was removed by centrifuging the tubes at 2,000 &#215; g for 10 min in a refrigerated centrifuge. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63695/63695fig03.jpg" /> 
Figure 3: Flow cytometric profiling of neutrophil population in peritoneal lavage fluid. Peritoneal lavage fluid was collected 16 h post infection. The peritoneal cells were stained with FITC-tagged anti-LY6G antibody. Septic mice showed increased infiltration of neutrophils in the peritoneal cavity. Abbreviations: FITC = fluorescein isothiocyanate; LY6G = lymphocyte antigen 6 complex locus G6D; SSC-A = side-scatter area. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

characterizing-salmonella-typhimurium-induced-septic-peritonitis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.2K Views.  Indian Institute of Science. This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Video: Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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Chapters in this video

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Measuring Local Anaphylaxis in Mice

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Quantification of Atherosclerosis in Mice

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Chronic Salmonella Infection Induced Intestinal Fibrosis